This application is to extend a MERIT award to study RNA synthesis in minus-strand RNA viruses. Nonsegmented negative-sense (NNS) RNA viruses include some of the most significant human pathogens that are an ongoing threat to US public health. For measles, mumps and rabies there are licensed vaccines, but for most NNS RNA viruses there are no vaccines and no antiviral drugs. Our long-term objective is to understand the mechanisms by which the replication machinery of vesicular stomatitis virus (VSV), a prototype of the NNS RNA viruses, functions. VSV is the ideal choice for such studies because it is the only NNS RNA virus for which robust transcription can be reconstituted in vitro from purified recombinant components. The catalytic core of the RNA synthesis machinery is a 241 kDa large protein (L) that contains an RNA dependent RNA polymerase (RdRP), a polyribonucleotidyltransferase (PRNTase) that caps the mRNA, and a dual specificity mRNA cap methyltransferase (MTase). During mRNA synthesis, those activities are coordinated so that the nascent mRNA is capped, methylated and polyadenylated. Although L contains all the enzymatic activities for RNA synthesis, it requires a 29 kDa phosphoprotein (P) that bridges interactions between L and the nucleocapsid protein (N) that completely coats the RNA template. Since the last competing renewal of this grant, we have obtained an atomic model of the VSV L protein. That structure profoundly changes our understanding of the RNA synthesis machinery of the NNS RNA viruses revealing that dynamic inter domain rearrangements in L protein must occur during RNA synthesis, identifying an intimate linkage between capping and RNA synthesis and identifying key P-L interactions that likely facilitate the inter domain rearrangements. Our underlying hypothesis is that the catalytic activities of L in RNA polymerization, mRNA cap addition and cap methylation which reside within structurally separate domains are coordinated by the presence of the P and the template associated N to regulate their activities during mRNA synthesis, and to downregulate them during assembly by complex formation with the viral matrix protein (M). A major gap in understanding the machinery of RNA synthesis is the existing structures of L likely represent the pre-initiation form of the transcriptase. We have in hand an interpretable density map of a rabies virus L-P complex from cryo EM. During the next funding period, we will continue to use cryo electron microscopy (EM), negative-stain EM, X-ray crystallography, in vitro biochemistry of polymerase and molecular virology to provide unique structural and functional insights into VSV and rabies virus polymerases during distinct stages of RNA synthesis and assembly. The successful completion of this work will provide an atomic level structure of an NNS RNA virus polymerase complex and new mechanistic insights into the function and regulation of this RNA synthesis machine during transcription, replication and assembly. Those results may aid efforts toward rational attenuation of NNS RNA viruses for vaccine purposes, and the development of antiviral therapeutics. RELEVANCE (See instructions): The L polymerase protein of nonsegmented negative-strand (NNS) RNA viruses contains an RNA dependent RNA polymerase and a suite of unconventional mRNA capping enzymes including a polyribonucleotidyltransferase and a dual-specificity cap methyltransfease. Understanding how those activities are controlled to ensure that the viral genome is expressed is of intrinsic interest and has the potential to impact the development of antiviral drugs and live attenuated vaccines. Here we will compare structural and functional insights into this protein for a prototype NNS RNA virus, vesicular stomatitis virus and the human pathogen rabies.